Taxol is a naturally occurring compound originally isolated from the stem bark of the Western Yew, Taxus brevifolia (Wani et al., J. Am. Chem. Soc., vol. 93 (1971), pg. 2325). Taxol is an important chemotherapeutic agent for the treatment of human ovarian, breast and lung cancers and is considered as a promising therapeutic agent for the treatment of other human cancers (Goldspiel, “Clinical Overview of the Taxanes”, Pharmacotherapy, vol. 17 (1997), pg. 110S and McGuire and Rowinsky, “Paclitaxel in Cancer Treatment”, Marcel Dekker, Inc., New York, 1995). Taxanes such as taxol, cephalomannine, baccatin III, and their 10-deacylated and 7-xylosyl derivatives are characterized by a highly complicated diterpene skeleton. These chemically similar compounds are frequently found together in complex extracts of different parts of yews. The isolation of individual taxanes from such extracts generally requires reverse phase chromatographic methods using expensive reverse phase silica based gel matrices, or alternatively, using silica gel or alumina matrices with very slow gradient elution systems (Snader, “Isolation and Detection,” in Taxol-Science and Applications, M. Suffness, ed., CRC Press, Boca Raton, Fla. 1995, pp. 277-286). Low isolation yields of desired compounds, the high costs of the chromatographic resins and/or regeneration of these resins are often unsatisfactory and contribute to high production costs.
The first isolation of taxol used a methanol extract from the bark of T. brevifolia followed by a series of chromatographic steps using FLORISIL®, SEPHADEX LH-20® and silica gel (Wani et al., J. Am. Chem. Soc., vol. 93 (1971), pg. 2325). Miller et al., J. Org. Chem., vol. 46 (1981), pg. 1469, describes the isolation of taxol and cephalomannine by ethanol extraction of the needles, twigs and roots of yews followed by counter current distribution, reverse phase chromatography and silica gel chromatography.
Production of taxol and other taxanes from yew biomass by prior art methods has been hindered by the often high levels of impurities in initial extracts. Various fractionation and filtration procedures have been used to enrich extracts before further purification. These include methods described in Rao WO 92/07842; Nair WO 94/13827; Elsohley et al. WO 92/18492; Carver et al. U.S. Pat. No. 5,281,727, issued Jan. 25, 1994; Pandey and Yankov, U.S. Pat. No. 5,654,448, issued Aug. 5, 1987; Cociancich and Pace, U.S. Pat. No. 5,744,333, issued Apr. 28, 1998; Liu U.S. Pat. No. 5,969,165, issued Oct. 19, 1999; and Snader, “Isolation and Detection,” in Taxol-Science and Applications, M. Suffness, ed., CRC Press, Boca Raton, Fla. 1995, pp. 277-286. Features shared by all these methods are an initial organic solvent extraction of the biomass, followed by one or more liquid:liquid partitioning steps to remove impurities, followed in turn by multiple chromatographic purification steps. Due to the low content of taxol in yew biomass (0.05-0.001% of dry weight), extraordinarily large amounts of organic solvents such as chloroform, methylene chloride and benzene are used in the extraction and partitioning procedures. These solvents are generally toxic and require costly emission control and waste management systems. In addition, the partitioning steps are highly prone to the formation of emulsions that seriously jeopardize the partition efficiency.
Nair, in WO 94/13827 and Liu in U.S. Pat. No. 5,969,165 disclose that treatment of a crude extract with activated charcoal as an essential step to avoid problems in downstream chromatographic separation steps. After removing the charcoal, the resulting clarified solution was fractionated either by precipitating the taxanes or by liquid:liquid partitioning steps to remove water soluble impurities prior to chromatography. Activated charcoal can absorb substantial amounts of the desired taxanes along with the impurities, and the liquid:liquid partitioning step can involve toxic solvents. This significantly reduces the process yield and can increase costs to isolate individual taxane compounds.
All of these drawbacks make the production of taxol and other taxanes from needles and twigs economically less attractive than the wholesale extraction from bark where the concentration of taxol is high and the concentration of problematic impurities tends to be relatively lower. However, there is concern that the isolation of taxol from yew bark will not be able to provide sufficient quantities for all cancer patients and would endanger the survival of natural yew. It is therefore desirable to develop high yield methods of purification of taxol and other taxanes from renewable sources of natural or cultivated yew biomass (Witherup et al., J. Nat. Prod., vol. 53 (1990), pg. 1249).
The present invention overcomes the above-mentioned drawbacks and provides additional benefits and advantages. Advantages include providing a process for the high yield extraction and purification of taxol and other taxanes from yew biomass. Other benefits include eliminating costly partitioning process, thus dramatically reducing the use of toxic organic solvents and expensive silica-based reverse phase chromatographic matrices.